Hexanucleotide (GGGGCC) repeat expansions in C9orf72 are the leading genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In this proposal, we seek to identify new therapeutic targets by using a genetically diverse mouse model of C9-FTD/ALS. In this study design, we will be using adeno-associated virus (AAV) to drive mutant C9-repeat expression (AAV-149R) in a panel of Collaborative Cross (CC) recombinant inbred mice, as well as Diversity Outbred (DO) mice produced from random repeated outcrossing of CC strains. Collectively, the CC and DO mouse populations offer high mapping resolution and broad allelic diversity, carrying 45 million SNPs and structural variants. This provides a unique opportunity to discover new genes that regulate C9-repeat toxicity in vivo. These modifiers will be compared to a parallel study of CC/DO mice that will be injected with AAV-TauP301L to determine modifiers of tau toxicity. In Aim 1 we will use CC mice to identify strains that are sensitive or resistant to C9 or tau toxicity while in Aim 2 we will use the DO mice to finely map these potential modifiers. In Aim 3 we will perform single- cell RNAseq studies from AAV-149R and AAV-TauP301L mice at stages of pre-symptomatic, as well as moderate and severe disease to determine the evolution of the brain response to these pathologies at the individual cell level. Also in Aim 3, we will perform single-nucleus RNAseq on postmortem human tissue from healthy controls, c9-ALS, c9-FTD, and FTLD-TauP301L from frontal cortex. Top hits from these datasets will be used to prioritize candidate genes/loci from Aims 1 and 2, and followed up with validation studies on postmortem tissue at the histological and biochemical level. At the conclusion of this comprehensive study, we hope to have identified numerous modifiers of both diseases that can be followed up for more in depth functional studies by our laboratories or the broader scientific community. It is likely that some of these genetic modifiers could also impact other neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, etc. and could be followed up by targeted testing in those disease models.